This invention provides for the cloning and expression of the human Macrophage Inflammatory Protein-1.alpha. (MIP-1.alpha.)/RANTES Receptor. This receptor binds two cytokines MIP-1.alpha. and RANTES which are pro-inflammatory cytokines. The receptor is useful for assaying the levels of these cytokines in biological specimens. These cytokines play key roles in the inflammatory processes afflicting man.
The chemokine .beta. division of the platelet factor 4 superfamily is comprised of at least six distinct cytokines that regulate trafficking of phagocytes and lymphocytes in mammalian species; at least one of these, MIP-1.alpha. also possesses anti-proliferative activity for hematopoietic stem cells. MIP-1.alpha. and the related .beta. chemokine, RANTES, induce transient alterations in intracellular Ca.sup.2+ concentration in neutrophils that can be reciprocally and specifically desensitized, suggesting a common receptor. This invention provides both the cDNA and the gene for this receptor. The receptor is a member of the G protein-coupled receptor superfamily. It has .sup..about. 33% amino acid identity with receptors for the .alpha. chemokine, interleukin-8, and may be the human homologue of the product of US28, an open reading frame of human cytomegalovirus.
The platelet factor 4 cytokine superfamily is comprised of structurally and functionally related 8-10 kilodalton peptides that are the products of distinct genes clustered on human chromosomes 4 and 17. The superfamily was named after the first member to be characterized. These peptides have been collectively designated as "chemokines" because of their hybrid activities: they regulate the trafficking and activation of lymphocytes and phagocytes of the mammalian immune system, but in addition, some are able to regulate the proliferative potential of hematopoietic progenitor cells, endothelial cells, and certain types of transformed cells (for reviews see Wolpe and Cerami, FASEB J. 3, 2565-2573 (1989); Oppenheim et al., Immunol. 9, 617-648 (1991); Schall, Cytokine 3, 165-183 (1991)). Thus, the chemokines are thought to play an important role in host defense against infection, in the pathogenesis of chronic inflammatory disorders, and in wound healing.
A structural signature common to all chemokines is the presence of four conserved cysteine residues. The chemokines can be divided into two groups, .alpha. and .beta., by the arrangement of the first two of these cysteines. Members of the chemokine .alpha. group all possess a single amino acid of variable identity interposed between the first two cysteines, the so-called "C--X--C" motif. The human chemokine .alpha. family includes platelet factor 4, interleukin-8/neutrophil activating peptide-1 (IL-8), GRO.alpha./melanoma growth stimulatory activity (GRO.alpha./MGSA), neutrophil activating peptide-2 (NAP-2), and other less well-characterized molecules. In general, members of this group are potent chemoattractants for neutrophils in vivo and in vitro; IL-8 and platelet factor 4 also regulate angiogenesis. (Koch et al., Science 258, 1798-1801 (1992); Maione et al., Science 247, 77-79 (1990)). Two human chemokine .alpha. receptors, designated IL-8 receptors A and B, have been identified; their structures are 77% identical at the amino acid level. IL-8 receptor B is a receptor for IL-8, NAP-2 and GRO.alpha./MGSA (Murphy and Tiffany, Science 253, 1280-1283 (1991); Lee et al., J. Biol. Chem. 267, 16283-16287 (1992)) whereas IL-8 receptor A is more selective for IL-8 (Holmes et al., Science 253, 1278-1280 (1991); Lee et al., J. Biol. Chem. 267, 16283-16287 (1992)). Signal transduction by both receptor subtypes leads to a rapid rise in the intracellular concentration of Ca.sup.2+.
In contrast, the first two conserved cysteines of all members of the chemokine .beta. group are adjacent, the "C--C" motif. Members of this group attract and activate neutrophils, eosinophils, monocytes, macrophages and lymphocytes with variable selectivity. The human chemokine .beta. family includes MIP-1.alpha. (Macrophage Inflammatory Protein-1.alpha.), MIP-1.beta., RANTES (Regulated on Activation, Normal T Expressed and Secreted), MCP-1 (Monocyte Chemoattractant Protein-1), MCP-2, MCP-3 and I-309. Of these, the biological properties of MIP-1.alpha., RANTES and MCP-1 have been the most characterized.
Murine MIP-1 was originally purified from supernatants of endotoxin-activated macrophages as a complex of MIP-1.alpha. and MIP-1.beta. which are 67% identical in amino acid sequence (Wolpe et al., J. Exp. Med. 167, 570-581 (1988); Davatelis et al., J. Exp. Med. 167, 1939-1944 (1988); Sherry et al., J. Exp. Med. 168, 2251-2259 (1988)).
In addition to being a chemoattractant for neutrophils (Wolpe et al., J. Exp. Med. 167, 570-581 (1988); Saukkonen et al., J. Exp. Med. 171, 439-448 (1990)), murine MIP-1 is a prostaglandin-independent endogenous pyrogen, has autocrine effects on macrophages, and may be involved in wound healing (Davatelis et al., Science 243, 1066-1068 (1989); Fahey et al., J. Immunol. 148, 2764-2769 (1992); Fahey et al., Cytokine 2, 92-98 (1990)). Many if not all of these effects are due to MIP-1.alpha.. In addition, MIP-1.alpha. suppresses proliferation of hematopoietic stem cells (Graham et al., Nature 344, 442-444 (1990); Broxmeyer et al., Blood 76, 1110-1116 (1990); Broxmeyer et al., J. Immunol. 147, 2586-2594 (1991); Dunlop et al., Blood 79, 2221-2225 (1992)). For this reason it has been suggested that MIP-1.alpha. may be a useful cytoprotective agent for clinical radiotherapy and chemotherapy of neoplastic disease (Dunlop et al., Blood 79, 2221-2225 (1992)). Limited functional information for MIP-1.alpha. using the human ligand and human targets is available. High and low affinity binding sites have been reported for human MIP-1.alpha. on the human myeloid cell line U937 (Yamamura et ai., Int. J. Hematol. 55, 131-137 (1992)); in contrast, a single class of binding sites for murine MIP-1.alpha. was detected on murine T cell and macrophage cell lines (Oh et al., J. Immunol. 147, 2978-2983 (1991)).
RANTES attracts eosinophils, monocytes and "memory" T cells (CD4.sup.+ /CD45RO.sup.+). It is a weak chemoattractant for neutrophils (Kameyoshi et ai., J. Exp. Med. 176, 587-592 (1992); Schall et al., Nature 347, 669-671 (1990); Schall, Cytokine 3, 165-183 (1991)). Characterization of binding sites for RANTES has not yet been reported. MCP-1 is a potent and specific chemoattractant and activating factor for monocytes (reviewed in Matsushima and Oppenheim, Cytokine 1, 2-13(1989)). Binding sites have been detected for MCP-1 on human monocytes (Yoshimura and Leonard, J. Immunol. 145, 292-297 (1990)).